1.4 - Eubacteria

Domain Bacteria

  • Prokaryotes with no nucleus; unicellular organisms.

  • All have peptidoglycan (PG) in their cell walls.

  • Size, shape, and arrangement vary widely; reproduce by binary fission.

  • Two main categories based on cell wall structure/staining: Gram-positive bacteria and Gram-negative bacteria. Also acid-fast bacteria (discussed in lecture 14).

  • Eubacteria terminology and diversity are foundational to understanding bacterial biology and taxonomy.

Domains of Life

  • Life is divided into three domains: Bacteria, Archaea, and Eukarya.

  • Bacteria and Archaea are prokaryotic; Eukarya are eukaryotic.

  • Unicellular vs multicellular organization:

    • Bacteria: unicellular

    • Archaea: unicellular

    • Eukarya: can be unicellular or multicellular

  • LUCA: Last Universal Common Ancestor, from which Bacteria, Archaea, and Eukarya diverged.

  • Representative groups:

    • Bacteria: Proteobacteria, Gram-positive bacteria, Cyanobacteria

    • Archaea: Euryarchaeota, Crenarchaeota

    • Eukarya: Fungi, Plants, Protists, Animals (amoebas, ciliates, slime molds, etc.)

  • Note on diversity: The spectrum of prokaryotes is vast; between 100,000 and 10,000,000 species are estimated, with about 12,000 described to date.

Bacterial Shapes and Arrangements

  • Shapes:

    • Bacilli (singular: bacillus) – rod-shaped

    • Cocci (singular: coccus) – spherical

    • Vibrio – curved rod

    • Coccobacillus – short, rounded rod

    • Spirochete – spiral

  • Arrangements:

    • Diplococci – pairs

    • Streptococci – chains

    • Staphylococci – clusters

  • Classification and morphology have historically guided taxonomy and helped in identification.

Morphology and Physiology in Classification

  • Historical approach (starting in the 1870s): classification based on morphology (shape, size, arrangement) and colony morphology (size, shape, color, biochemical reactions).

  • Before molecular methods, colonies could show 1 million to 100 million cells per colony.

  • Since the 1950s, genome sequencing has transformed classification, leading to reclassifications and name changes.

Bacterial Terminology

  • Species (biology): a group of organisms capable of exchanging genes or interbreeding.

  • Species (bacteriology): defined by genetic similarity, biochemical traits, and phenotypic criteria.

  • Bacterial strain: genetic variants within a species with unique characteristics (specific structures, proteins, or genes).

Species Examples in E. coli

  • E. coli and its diverse pathotypes:

    • EPEC: Enteropathogenic E. coli

    • EHEC: Enterohemorrhagic E. coli

    • ETEC: Enterotoxigenic E. coli

    • EAEC: Enteraggregative E. coli

    • EIEC: Enteroinvasive E. coli

    • DAEC: Diffusely aggregative E. coli

  • These illustrate genetic and phenotypic diversity within a single species.

Bacterial Cells Divide by Binary Fission

  • Prokaryotic reproduction is by binary fission.

  • Resulting progeny are genetically identical, assuming no mutations occur during replication.

  • Mechanism underlies rapid population growth in favorable conditions.

Bacterial Cell Structure: Overview of Common Structures

  • Core structures in many bacteria include:

    • Cell wall

    • Cell membrane

    • Glycocalyx (capsule or slime layer)

    • Flagella (motility) and Pili (attachment and genetic exchange)

    • Nucleoid (region containing the chromosome)

    • Plasmids (extrachromosomal DNA)

    • Ribosomes (protein synthesis)

    • Cytoplasm (cytosol)

  • Not all structures are present in every species.

Gram-Positive Bacteria: Cell Wall

  • Composition: Thick peptidoglycan (PG) layer with teichoic acids; associated with the cell membrane.

  • Function: Structural integrity and protection against mechanical stress and desiccation.

  • Key components: PG layers and teichoic acids.

  • Location of structures: Cell wall is external to the cell membrane; thick PG layer is a hallmark of GPB.

Gram-Positive Bacteria: Peptidoglycan and PBPs

  • Peptidoglycan (PG) is a mesh-like, rigid lattice around the cell.

  • PG is composed of long strands of sugars: NAM and NAG, linked by peptide cross-links.

  • The glycosidic bond between NAM and NAG is lysozyme-sensitive.

  • Enzymes called Penicillin-Binding Proteins (PBPs) create the cross-links in PG.

  • PBPs are targets of beta-lactam antibiotics (e.g., penicillin, amoxicillin).

  • Antimicrobial resistance can arise from alterations in PBPs (e.g., Methicillin-Resistant Staphylococcus aureus, MRSA).

Gram-Positive: Penicillin Action and Resistance

  • Penicillin and related beta-lactams inhibit PBPs, blocking cross-linking in PG and weakening the cell wall.

  • This weakening leads to cell lysis as the cell grows and expands.

  • Example of resistance: MRSA shows altered PBPs, reducing beta-lactam binding and effectiveness.

  • Visual: Penicillin disrupts cell wall synthesis during binary fission, leading to rupture.

Gram-Negative Bacteria: Cell Wall

  • Composition: Thin PG layer; outer membrane; periplasmic space; and an outer membrane containing LPS.

  • Function: Structural integrity and protection; enhanced defense against certain chemicals.

  • Organization: PG layer is thinner than in GPB; outer membrane provides an additional barrier.

  • Location: Outer membrane external to the PG layer.

LPS: Lipopolysaccharide in Gram-Negative Bacteria

  • Structure:

    • Lipid A: embedded in the outer membrane

    • Core polysaccharide: about 10 sugars

    • O-antigen: extended polysaccharide chains outward from the cell surface

  • Function:

    • Regulates transport of molecules and nutrients into/out of the cell

    • Provides protection against some host defenses

    • O-antigen is highly variable, helping bacteria evade immune detection

  • Endotoxin: LPS acts as an endotoxin; large amounts released upon bacterial cell lysis can trigger fever and septic shock.

  • Visual: Outer membrane with LPS, periplasmic space, and thin PG layer between the outer membrane and cytoplasmic membrane.

LPS Functions and Pathophysiology

  • LPS enables controlled permeability in Gram-negative bacteria and contributes to pathogenicity.

  • The O-antigen variability contributes to immune evasion.

  • LPS is an endotoxin that can cause systemic inflammatory responses when released in large amounts during infection.

How the Gram Stain Identifies Differences in Cell Wall Structure

  • Gram staining is a differential stain:

    • Gram-positive bacteria: Thick PG layer retains Crystal Violet; cells appear purple.

    • Gram-negative bacteria: Outer membrane + thin PG layer cause Crystal Violet to wash out; cells are counterstained pink by Safranin.

  • Stain steps are historically designed to differentiate based on cell wall architecture.

Gram Stain Examples and Visuals

  • Practical examples include controls and clinical specimens demonstrating GPB vs GNB staining patterns.

Surface Structures: Cell Membrane

  • Structure: Phospholipid bilayer.

  • Composition: Lipids and proteins.

  • Function: Regulates transport into and out of the cell.

  • Location: Internal boundary just inside the cell wall in Gram-positive bacteria and just inside the outer membrane in Gram-negative bacteria.

Surface Structures: Glycocalyx / Capsule

  • Glycocalyx (capsule) composition: Carbohydrates.

  • Function: Protection from environmental stresses; attachment to surfaces; can contribute to virulence in pathogenic bacteria.

  • Appearance: May appear as a slime layer or capsule around the cell.

Surface Appendages: Flagella and Pili

  • Flagellum (flagella):

    • Composition: Protein filament (pilin) attached to the cell envelope.

    • Function: Motility; propels the cell; can influence chemotaxis and movement.

    • Structure: Flagellar filament rotates, enabling movement; can alter speed and direction.

  • Pili (pilus):

    • Composition: Protein.

    • Function: Attachment to surfaces; genetic exchange through conjugation.

  • Figures referenced show typical flagellar and pili structures.

Bacterial Motility: Flagella in Action

  • If a single flagellum is attached to the substratum, the cell body may rotate/spin; flagella act like propellers.

  • E. coli examples and general motility metrics often cited in teaching materials.

  • Typical dimensions and motility speeds observed in experimental images and demonstrations.

Cytoplasmic Structures

  • Cytoplasm (cytosol): Aqueous interior of the cell where most biochemical reactions occur.

  • Nucleoid: Region containing the bacterial chromosome.

  • Chromosome: Usually a single circular chromosome (rare exceptions may exist).

Plasmids

  • Plasmids: Extrachromosomal circular DNA.

  • Function: Carry accessory genetic information; often non-essential but can confer survival advantages (e.g., multidrug resistance, MDR).

  • Visual: Small, circular DNA elements separate from the chromosome.

Ribosomes

  • Function: Protein synthesis for the cell.

  • Composition: RNA and protein.

  • Prokaryotic ribosomes: 70S, composed of two subunits: 50S (large) and 30S (small).

  • Clinical relevance: Common targets for antibiotics that inhibit bacterial protein synthesis.

Endospores

  • Endospores are produced by select Gram-positive species as a response to stress (nutrient limitation, heat, desiccation).

  • Features: Metabolically inactive, highly resistant to heat, drying, chemicals, and starvation.

  • Function: Long-term survival; can return to vegetative growth when conditions improve.

The Spectrum of Prokaryotes: Diversity and Taxonomy

  • The domains Bacteria and Archaea together harbor enormous diversity.

  • Estimated species range: between 100,000 and 10,000,000; described/described: about 12,000.

  • Figures in lectures illustrate the breadth of prokaryotic diversity and the central concept of a LUCA.

Take-Home Messages

  • Key shapes and arrangements to recognize in bacterial cells.

  • The usefulness of staining techniques like Gram staining for differentiating cell wall types.

  • Surface structures (cell membrane, glycocalyx, flagella, pili) and intracellular components (nucleoid, plasmids, ribosomes, cytoplasm).

  • Binary fission as the primary mode of prokaryotic reproduction.

  • Endospores as a survival strategy in certain Gram-positive bacteria.

  • The diversity of prokaryotes and their relevance to health, disease, and environmental biology.

Terminology (glossary concepts)

  • Bacteria / Eubacteria

  • Gram positive bacteria (GPB)

  • Gram negative bacteria (GNB)

  • Prokaryote / prokaryotic

  • Micron (µm)

  • Bacillus, Coccus, Vibrio, Coccobacillus, Spirochete

  • Diplococcus, Streptococcus, Staphylococcus

  • Binary fission

  • Peptidoglycan (PG)

  • Lipopolysaccharide (LPS)

  • Endotoxin

  • Differential stain

  • Conjugation

  • Plasmid

  • Ribosome

  • Endospore

  • Vegetative cell

  • Lysozyme (muramidase)

  • Penicillin Binding Proteins (PBPs)

  • Beta-lactams (penicillin, methicillin, amoxicillin, cephalosporins)

  • MRSA (Methicillin-Resistant Staphylococcus aureus)

  • LPS components: Lipid A, core polysaccharide, O-antigen

  • NAG (N-acetylglucosamine), NAM (N-acetylmuramic acid)

  • Proteobacteria, Cyanobacteria, Euryarchaeota, Crenarchaeota, etc.

Terminology Definitions

  • Bacteria / Eubacteria: Prokaryotes with no nucleus; unicellular organisms, all have peptidoglycan (PG) in their cell walls.

  • Gram-positive bacteria (GPB): Bacteria with a thick peptidoglycan layer that retains Crystal Violet stain, appearing purple.

  • Gram-negative bacteria (GNB): Bacteria with a thin peptidoglycan layer and an outer membrane; Crystal Violet washes out, and cells are counterstained pink by Safranin.

  • Prokaryote / prokaryotic: Organisms (Bacteria and Archaea) characterized by the absence of a membrane-bound nucleus and other organelles.

  • Micron (µm): A unit of length equal to one millionth of a meter (10^{-6}\text{ m}), used to measure microscopic organisms.

  • Bacillus: Rod-shaped bacterial cell.

  • Coccus: Spherical bacterial cell.

  • Vibrio: Curved rod-shaped bacterial cell.

  • Coccobacillus: Short, rounded rod-shaped bacterial cell.

  • Spirochete: Spiral-shaped bacterial cell.

  • Diplococcus: Spherical bacteria arranged in pairs.

  • Streptococcus: Spherical bacteria arranged in chains.

  • Staphylococcus: Spherical bacteria arranged in clusters.

  • Binary fission: The primary mode of prokaryotic reproduction, where a single cell divides into two genetically identical daughter cells.

  • Peptidoglycan (PG): A mesh-like, rigid lattice composed of NAM and NAG sugars linked by peptide cross-links, forming the structural component of bacterial cell walls.

  • Lipopolysaccharide (LPS): A molecule composed of Lipid A, core polysaccharide, and O-antigen, found in the outer membrane of Gram-negative bacteria; acts as an endotoxin.

  • Endotoxin: A toxic component (LPS) released from Gram-negative bacteria upon cell lysis, capable of triggering fever and septic shock.

  • Differential stain: A staining technique (like Gram stain) that differentiates bacteria based on structural differences, typically in their cell walls.

  • Conjugation: A process of genetic exchange in bacteria, often mediated by pili, where DNA is transferred from one bacterium to another.

  • Plasmid: Extrachromosomal circular DNA molecules in bacteria that carry accessory genetic information, often conferring survival advantages like multidrug resistance.

  • Ribosome: Cellular structures composed of RNA and protein, responsible for protein synthesis in the cell (prokaryotic ribosomes are 70S).

  • Endospore: A highly resistant, metabolically inactive dormant structure produced by select Gram-positive species as a survival strategy against harsh conditions.

  • Vegetative cell: A metabolically active bacterial cell capable of growth and reproduction, in contrast to an endospore.

  • Lysozyme (muramidase): An enzyme that targets the glycosidic bond between NAM and NAG in peptidoglycan, leading to cell wall degradation.

  • Penicillin Binding Proteins (PBPs): Enzymes that create the cross-links in peptidoglycan; targets of beta-lactam antibiotics.

  • Beta-lactams (penicillin, methicillin, amoxicillin, cephalosporins): A class of antibiotics that inhibit Penicillin-Binding Proteins, blocking peptidoglycan cross-linking and weakening the bacterial cell wall.

  • MRSA (Methicillin-Resistant Staphylococcus aureus): A strain of Staphylococcus aureus that has altered PBPs, leading to resistance against beta-lactam antibiotics.

  • LPS components:

    • Lipid A: The lipid portion of LPS embedded in the outer membrane, responsible for endotoxin activity.

    • Core polysaccharide: A sugar chain (about 10 sugars) connecting Lipid A to the O-antigen.

    • O-antigen: An extended polysaccharide chain projecting outward from the cell surface; highly variable and contributes to immune evasion.

  • NAG (N-acetylglucosamine): One of the two alternating sugar components of peptidoglycan.

  • NAM (N-acetylmuramic acid): One of the two alternating sugar components of peptidoglycan.

  • Proteobacteria, Cyanobacteria, Euryarchaeota, Crenarchaeota, etc. Representitive groups of Bacteria and Archaea, illustrating prokaryotic diversity.

Basic Description of a Prokaryotic Cell

Prokaryotic cells (Domains Bacteria and Archaea) are unicellular organisms that lack a membrane-bound nucleus and other membrane-bound organelles. They contain a nucleoid region where their single circular chromosome is located, ribosomes (70S) for protein synthesis, a cytoplasm, a cell membrane, and typically a cell wall. Many also possess accessory structures like glycocalyx, flagella, and pili.

Various Shapes and Arrangements of Prokaryotes

  • Shapes:

    • Bacilli: Rod-shaped

    • Cocci: Spherical

    • Vibrio: Curved rod

    • Coccobacillus: Short, rounded rod

    • Spirochete: Spiral

  • Arrangements:

    • Diplococci: Pairs of spherical cells

    • Streptococci: Chains of spherical cells

    • Staphylococci: Clusters of spherical cells

Steps in Gram Staining and Structural Reason for Staining Differences

Gram staining is a differential stain used to categorize bacteria based on their cell wall structure:

  1. Crystal Violet (primary stain): Applied to bacterial cells; both Gram-positive and Gram-negative cells absorb the purple dye.

  2. Iodine (mordant): Forms a complex with crystal violet, trapping it within the cell.

  3. Alcohol/Acetone (decolorizer):

    • Gram-positive bacteria: The thick peptidoglycan layer dehydrates and shrinks, trapping the Crystal Violet-iodine complex. Cells remain purple.

    • Gram-negative bacteria: The alcohol dissolves the outer membrane, and the thin peptidoglycan layer cannot retain the Crystal Violet-iodine complex, allowing the dye to wash out. Cells become colorless.

  4. Safranin (counterstain):

    • Gram-positive bacteria: Cells remain purple.

    • Gram-negative bacteria: Cells are stained pink/red by the safranin.

Comparison of Gram-Positive and Gram-Negative Cell Walls

Feature

Gram-Positive Bacteria

Gram-Negative Bacteria

Peptidoglycan (PG)

Thick layer

Thin layer

Outer Membrane

Absent

Present (external to PG layer)

Teichoic Acids

Present (integrated into PG layer)

Absent

LPS

Absent

Present (in outer membrane)

Periplasmic Space

Absent or very small

Present (between outer membrane and cytoplasmic membrane, surrounding PG)

Stain Result

Purple

Pink/Red

Protection

Structural integrity, protection against mechanical stress and desiccation

Enhanced defense against certain chemicals, additional barrier

Antimicrobials Targeting the Gram-Positive Cell Wall

Lysozyme and beta-lactam antibiotics (e.g., penicillin) target the Gram-positive cell wall because their thick peptidoglycan (PG) layer is critical for structural integrity and is readily accessible.

  • Lysozyme targets the (\beta-1,4) glycosidic bond between NAM and NAG sugars in the PG backbone, effectively breaking down the mesh-like structure.

  • Beta-lactams (like penicillin) inhibit Penicillin-Binding Proteins (PBPs), which are enzymes responsible for creating the peptide cross-links that give PG its rigidity. By blocking these cross-links, beta-lactams weaken the cell wall. During binary fission, the cell expands, and the weakened wall leads to cell lysis.

Structure, Composition, and Function of Bacterial Cell Components

  • Bacterial Cell Wall:

    • Structure/Composition: Mesh-like layer made of peptidoglycan (NAM and NAG sugars cross-linked by peptides). Gram-positive bacteria have a thick PG layer with teichoic acids. Gram-negative bacteria have a thin PG layer and an outer membrane containing LPS.

    • Function: Provides structural integrity, maintains cell shape, and protects against osmotic lysis, mechanical stress, and desiccation.

  • Cell Membrane (Cytoplasmic Membrane):

    • Structure/Composition: Phospholipid bilayer with embedded proteins.

    • Function: Regulates selective transport of molecules and nutrients into and out of the cell; involved in energy generation (e.g., electron transport chain).

  • Flagella:

    • Structure/Composition: Long, helical protein filaments (made of pilin) attached to the cell envelope by a basal body and hook.

    • Function: Motility; functions like a propeller to move the cell through liquid environments. Enables chemotaxis (movement in response to chemical gradients).

  • Pili (Fimbriae):

    • Structure/Composition: Short, hair-like protein appendages.

    • Function: Attachment to surfaces (adhesion), formation of biofilms, and genetic exchange (conjugation) via sex pili.

  • Glycocalyx / Capsule:

    • Structure/Composition: An outermost layer composed of carbohydrates (polysaccharides).

    • Function: Protection from environmental stresses (e.g., desiccation), aids in attachment to surfaces, and can contribute to virulence by preventing phagocytosis by host immune cells.

  • Ribosomes:

    • Structure/Composition: Composed of ribosomal RNA (rRNA) and proteins, prokaryotic ribosomes are 70S (composed of a 50S large subunit and a 30S small subunit).

    • Function: Essential for protein synthesis, translating messenger RNA into polypeptide chains.

  • Plasmids:

    • Structure/Composition: Small, extrachromosomal, circular DNA molecules.

    • Function: Carry non-essential but often beneficial genes, such as those conferring antibiotic resistance (multidrug resistance, MDR), virulence factors, or metabolic capabilities.

Functions of LPS including as an Endotoxin

LPS (Lipopolysaccharide) is a crucial component of the outer membrane in Gram-negative bacteria with several functions:

  • Structure: Contributes to the structural integrity and stability of the outer membrane.

  • Controlled Permeability: Regulates the transport of molecules and nutrients into/out of the cell, acting as a selective barrier.

  • Immune Evasion: The highly variable O-antigen component helps bacteria evade immune detection by host antibodies.

  • Endotoxin Activity: The Lipid A component of LPS acts as an endotoxin. When Gram-negative bacterial cells lyse (e.g., during infection or antibiotic treatment), large amounts of Lipid A are released. This release can trigger strong systemic inflammatory responses in the host, leading to symptoms like fever, inflammation, and potentially life-threatening septic shock.

Function of Endospores

Endospores are dormant, highly resistant structures produced by certain Gram-positive bacteria (e.g., Bacillus, Clostridium) in response to unfavorable environmental conditions such as nutrient limitation, extreme temperatures, desiccation, or chemical exposure. Their primary function is long-term survival under harsh conditions. They are metabolically inactive and can remain viable for extended periods. When favorable conditions return, the endospore can germinate and develop into a metabolically active vegetative cell.

Binary Fission

Binary fission is the primary method of asexual reproduction in prokaryotic cells. It is a simple and rapid process involving:

  1. DNA Replication: The single circular bacterial chromosome replicates, starting from an origin of replication.

  2. Cell Elongation: The cell elongates, and the two replicated chromosomes move to opposite ends of the cell.

  3. Septum Formation: A septum (new cell wall and membrane) begins to form down the center of the elongated cell.

  4. Cell Division: The septum grows inward, eventually dividing the parent cell into two genetically identical daughter cells. This mechanism enables rapid population growth under favorable conditions.